RESUMEN
BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Gotosa/tratamiento farmacológico , Supresores de la Gota/uso terapéutico , Hiperalgesia/prevención & control , Proteínas Recombinantes de Fusión/uso terapéutico , Sinovitis/prevención & control , Animales , Artritis Experimental/complicaciones , Artritis Gotosa/complicaciones , Biomarcadores/metabolismo , Colchicina/uso terapéutico , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Hiperalgesia/etiología , Interleucina-1/antagonistas & inhibidores , Masculino , Ratones , Ratones Noqueados , Infiltración Neutrófila , Receptores Tipo I de Interleucina-1/deficiencia , Receptores Tipo I de Interleucina-1/genética , Transducción de Señal/fisiología , Sinovitis/etiología , Regulación hacia Arriba/efectos de los fármacos , Ácido ÚricoRESUMEN
Vascular endothelial growth factor (VEGF) is a protein factor which has been found to play a significant role in both normal and pathological states. Its role as an angiogenic factor is well-established. More recently, VEGF has been shown to protect neurons from cell death both in vivo and in vitro. While VEGF's potential as a protective factor has been demonstrated in hypoxia-ischemia, in vitro excitotoxicity, and motor neuron degeneration, its role in seizure-induced cell loss has received little attention. A potential role in seizures is suggested by Newton et al.'s [Newton SS, Collier EF, Hunsberger J, Adams D, Terwilliger R, Selvanayagam E, Duman RS (2003) Gene profile of electroconvulsive seizures: Induction of neurotrophic and angiogenic factors. J Neurosci 23:10841-10851] finding that VEGF mRNA increases in areas of the brain that are susceptible to cell loss after electroconvulsive-shock induced seizures. Because a linear relationship does not always exist between expression of mRNA and protein, we investigated whether VEGF protein expression increased after pilocarpine-induced status epilepticus. In addition, we administered exogenous VEGF in one experiment and blocked endogenous VEGF in another to determine whether VEGF exerts a neuroprotective effect against status epilepticus-induced cell loss in one vulnerable brain region, the rat hippocampus. Our data revealed that VEGF is dramatically up-regulated in neurons and glia in hippocampus, thalamus, amygdala, and neocortex 24 h after status epilepticus. VEGF induced significant preservation of hippocampal neurons, suggesting that VEGF may play a neuroprotective role following status epilepticus.
Asunto(s)
Hipocampo/metabolismo , Hipocampo/patología , Neuronas/metabolismo , Neuronas/patología , Convulsiones/metabolismo , Convulsiones/patología , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/ultraestructura , Muerte Celular/fisiología , Convulsivantes , Ensayo de Inmunoadsorción Enzimática , Hipocampo/citología , Inmunohistoquímica , Técnicas In Vitro , Bombas de Infusión Implantables , Masculino , Fármacos Neuroprotectores , Pilocarpina , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Estado Epiléptico/inducido químicamente , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The concept that tumors can be controlled by directly targeting their vascular supply has finally come of age, because clinical trials using a humanized monoclonal antibody that blocks VEGF have demonstrated exciting efficacy in cancer patients, as well as in vascular eye diseases that can lead to blindness. However, data suggest that these current regimens may not provide complete VEGF inhibition and, thus, that the maximum therapeutic potential of VEGF blockade has not yet been achieved. We describe the status of a very potent and high-affinity VEGF blocker, termed the VEGF Trap, that may provide the opportunity to maximize the potential of VEGF blockade in cancer as well as in vascular eye diseases. We also describe use of the VEGF Trap as a research tool, when coupled to high-throughput mouse genetics approaches such as VelociGene that can be exploited in strategies to discover and validate the next generation of angiogenesis targets.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Oftalmopatías/terapia , Neoplasias/irrigación sanguínea , Neoplasias/terapia , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Angiopoyetinas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Bevacizumab , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/terapia , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Factor A de Crecimiento Endotelial Vascular/genéticaAsunto(s)
Inductores de la Angiogénesis/fisiología , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/terapia , Glicoproteínas de Membrana/fisiología , Angiopoyetina 1 , Animales , Factores de Crecimiento Endotelial/fisiología , Humanos , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Modelos Biológicos , Músculo Liso Vascular/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor TIE-2 , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularAsunto(s)
Inductores de la Angiogénesis/fisiología , Factores de Crecimiento Endotelial/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Glicoproteínas de Membrana/fisiología , Neovascularización Patológica , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas , Inductores de la Angiogénesis/genética , Angiopoyetina 1 , Angiopoyetina 2 , Animales , Permeabilidad Capilar , Ligandos , Sistema Linfático/crecimiento & desarrollo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The possibility of stimulating or inhibiting paracrine factors regulating angiogenesis may lead to new approaches for the treatment of pathological conditions of the female reproductive tract. We examined the effects of a clinical candidate, a soluble truncated form of the Flt-1 receptor, vascular endothelial growth factor trap(A40) (VEGF trap), in a primate model to determine its ability to prevent the onset of luteal angiogenesis or intervene with the on-going process. Marmosets were treated from the day of ovulation until luteal day 3 (prevention regimen) or on luteal day 3 for 1 day (intervention regimen). Effects of VEGF inhibition were studied by obtaining a proliferation index using bromodeoxyuridine incorporation, quantifying endothelial cell area using CD31, and assessing luteal function by plasma progesterone. After both treatments, intense luteal endothelial proliferation was suppressed, a concomitant decrease in endothelial cell area confirmed the inhibition of vascular development, and a marked fall in plasma progesterone levels showed that luteal function was compromised. In situ hybridization was used to localize and quantify compensatory effects on the expression of angiogenic genes. VEGF messenger ribonucleic acid (mRNA) expression in luteal cells was increased, whereas expression of its receptor, Flt, was decreased. Inhibition of VEGF resulted in localized increased expression of angiopoietin-2 mRNA and its receptor, Tie-2. The results show that the VEGF trap can prevent luteal angiogenesis and inhibit the established process with resultant suppression of luteal function. Luteal Flt mRNA expression is dependent upon VEGF, and VEGF inhibition results in abortive increases in expression of VEGF, angiopoietin-2, and Tie-2.
Asunto(s)
Cuerpo Lúteo/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Proto-Oncogénicas/farmacología , Proteínas Tirosina Quinasas Receptoras/farmacología , Angiopoyetina 2 , Animales , Bromodesoxiuridina/análisis , Bromodesoxiuridina/metabolismo , Callithrix , División Celular , Cuerpo Lúteo/fisiología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Femenino , Fragmentos Fc de Inmunoglobulinas/genética , Hibridación in Situ , Fase Luteínica/efectos de los fármacos , Linfocinas/antagonistas & inhibidores , Linfocinas/genética , Tamaño de los Órganos , Ovario/anatomía & histología , Ovulación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Progesterona/sangre , Proteínas/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
A recent explosion in newly discovered vascular growth factors has coincided with exploitation of powerful new genetic approaches for studying vascular development. An emerging rule is that all of these factors must be used in perfect harmony to form functional vessels. These new findings also demand re-evaluation of therapeutic efforts aimed at regulating blood vessel growth in ischaemia, cancer and other pathological settings.
Asunto(s)
Factores de Crecimiento Endotelial/fisiología , Linfocinas/fisiología , Neovascularización Fisiológica , Angiopoyetina 2 , Animales , Factores de Crecimiento Endotelial/genética , Efrina-B2 , Linfocinas/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Neovascularización Patológica , Neovascularización Fisiológica/efectos de los fármacos , Proteínas/fisiología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/fisiología , Receptores TIE , Receptores de Factores de Crecimiento Endotelial Vascular , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents.
Asunto(s)
Glicoproteínas de Membrana/farmacología , Piel/irrigación sanguínea , Enfermedades Vasculares/tratamiento farmacológico , Angiopoyetina 1 , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Transformada , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/farmacología , Vectores Genéticos , Células HeLa , Humanos , Linfocinas/genética , Linfocinas/farmacología , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Ratones Transgénicos , Proteínas Recombinantes/farmacología , Piel/patología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Transgenic mice overexpressing brain-derived neurotrophic factor from the beta-actin promoter were tested for behavioral, gross anatomical and physiological abnormalities. Brain-derived neurotrophic factor messenger RNA overexpression was widespread throughout brain. Overexpression declined with age, such that levels of overexpression decreased sharply by nine months. Brain-derived neurotrophic factor transgenic mice had no gross deformities or behavioral abnormalities. However, they showed a significant passive avoidance deficit. This deficit was dependent on continued overexpression, and resolved with age as brain-derived neurotrophic factor transcripts decreased. In addition, the brain-derived neurotrophic factor transgenic mice showed increased seizure severity in response to kainic acid. Hippocampal slices from brain-derived neurotrophic factor transgenic mice showed hyperexcitability in area CA3 and entorhinal cortex, but not in dentate gyrus. Finally, area CA1 long-term potentiation was disrupted, indicating abnormal plasticity. Our data suggest that overexpression of brain-derived neurotrophic factor in the brain can interfere with normal brain function by causing learning impairments and increased excitability. The results also support the hypothesis that excess brain-derived neurotrophic factor could be pro-convulsant in the limbic system.
Asunto(s)
Reacción de Prevención/fisiología , Factor Neurotrófico Derivado del Encéfalo/genética , Giro Dentado/fisiopatología , Corteza Entorrinal/fisiopatología , Epilepsia/fisiopatología , Factores de Edad , Animales , Conducta Animal/fisiología , Northern Blotting , Química Encefálica/genética , Electrofisiología , Epilepsia/inducido químicamente , Agonistas de Aminoácidos Excitadores , Expresión Génica/fisiología , Calor , Hibridación in Situ , Ácido Kaínico , Potenciación a Largo Plazo/fisiología , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Técnicas de Cultivo de Órganos , Umbral del Dolor , ARN Mensajero/análisis , Natación , TransgenesRESUMEN
Fusion proteins comprising the Fc domain of human IgG and extracellular domains of receptor tyrosine kinases can neutralize the activity of their cognate ligands when administered in molar excess. We have generated a fusion protein using the ectodomain of TrkB (TrkB-Fc). Although the ability of TrkB-Fc to neutralize the activity of brain-derived neurotrophic factor (BDNF) in vitro has been demonstrated, there have been no conclusive demonstrations of its ability to neutralize the activity of BDNF in vivo. We co-infused TrkB-Fc with BDNF into the cortex and hippocampus of adult rats to determine whether TrkB-Fc would interfere with the ability of BDNF to upregulate neuropeptide Y (NPY). We report here that rather than neutralizing the activity of exogenous BDNF, co-infusion with the TrkB-Fc fusion protein greatly increased the volume of tissue in which neuropeptide Y immunostaining was upregulated. In addition, TrkB-Fc greatly enhanced BDNF's distribution through adult brain parenchyma. TrkB-Fc also markedly increased the otherwise limited diffusion of BDNF into brain parenchyma following intraventricular infusion. These results show that rather than neutralizing or sequestering BDNF, the TrkB-Fc, at close to molar equivalence to BDNF, can function as a carrier for BDNF and thus enhance the delivery or penetration of this polypeptide into the brain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacocinética , Encéfalo/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Fc/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Animales , Encéfalo/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Ensayo de Inmunoadsorción Enzimática , Hipocampo/metabolismo , Humanos , Inyecciones Intraventriculares , Masculino , Neuropéptido Y/biosíntesis , Neuropéptido Y/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinética , Spodoptera/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Systemic administration of the excitotoxin kainic acid to adult rats results in a well defined pattern of loss of the CA1 and CA3 pyramidal neurons of the hippocampus. Prior to this neuronal loss, brain-derived neurotrophic factor (BDNF) mRNA is substantially increased. We show here that BDNF protein is increased after excitotoxic insult in specific areas of the hippocampus, reaching maximal levels 24 h after the insult. BDNF protein levels in the hippocampus increase in direct relation to the severity of seizure. Up to 7 days after injection of kainic acid, levels of full-length TrkB protein were unchanged, whereas levels of truncated TrkB protein were significantly increased by 12 h. To determine whether elevations in BDNF protein levels are potentially beneficial to hippocampal neurons exposed to an excitotoxic stress, we infused exogenous BDNF prior to and during the period of neuronal death caused by kainic acid. We find that administration of high levels of exogenous BDNF does not affect severity of seizure, but does in fact, exacerbate the injury caused by kainic acid, specifically to CA3 pyramidal neurons. Although there was a trend toward sparing of CA1 pyramidal neurons on the side infused with BDNF, this was not significant. In the same paradigm, infusion of exogenous NT-3 had no effect.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/farmacología , Regulación de la Expresión Génica , Hipocampo/metabolismo , Ácido Kaínico/toxicidad , Células Piramidales/patología , Animales , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/patología , Cinética , Masculino , Factores de Crecimiento Nervioso/biosíntesis , Fármacos Neuroprotectores , Neurotoxinas/toxicidad , Neurotrofina 3 , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/metabolismo , Convulsiones/inducido químicamente , Convulsiones/patología , Convulsiones/fisiopatología , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Exposure of embryonic CNS neurons to BDNF in vitro causes down-regulation of TrkB protein and mRNA, and an attenuation of functional responses to acute neurotrophin stimulation. In order to investigate ligand-mediated regulation of TrkB in vivo, we infused BDNF into the midbrain, near the periaquaductal grey-dorsal raphe (PAG-DR), or into the olfactory bulb of adult rats. Midbrain infusion of BDNF produced analgesia that was sustained for the duration of BDNF delivery. Analysis of TrkB receptor levels revealed that at the point when the maximal analgesic effect of BDNF was obtained, there was a concommitant 75% decrease in full-length TrkB protein at the infusion site. After discontinuation of infusion, levels of TrkB recovered toward base line. Interestingly, TrkB protein levels were not accompanied by decreased trkB mRNA levels. To determine if BDNF infusion decreased TrkB protein levels in other brain areas and whether trkB mRNA might be down-regulated in the cell bodies of neurons projecting to the infusion site, we infused BDNF into the olfactory bulb. Following a 12-day infusion of BDNF, TrkB protein levels decreased within the bulb to a similar extent as in the PAG-DR. This decrease in receptor protein, however, was not accompanied by decreased trkB mRNA levels in the olfactory cortex, which is afferent to the bulb. Taken together, our data suggest that decreases in TrkB receptor protein at the site of BDNF infusions in the adult brain represent receptor turnover, but this is not associated with altered expression of trkB mRNA or attenuation of the pharmacological effects of BDNF.
Asunto(s)
Química Encefálica/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Receptores de Factor de Crecimiento Nervioso/genética , Factores de Edad , Animales , Conducta Animal/efectos de los fármacos , Transporte Biológico/fisiología , Western Blotting , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Mesencéfalo/química , Mesencéfalo/citología , Neuronas/química , Neuronas/citología , Nociceptores/efectos de los fármacos , Nociceptores/fisiología , Bulbo Olfatorio/química , Bulbo Olfatorio/citología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal Trkb protein does not appear to be just a development phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro, this leads to long-term functional desensitization to BDNF, NT-3 and NT-4/5, as well as down-regulation of trkB and trkC mRNA.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Hipocampo/efectos de los fármacos , Factores de Crecimiento Nervioso/fisiología , Neuronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados , Regulación hacia Abajo , Hipocampo/citología , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Factores de Crecimiento Nervioso/farmacología , Neurotrofina 3 , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
Neurotrophic factors have been shown to ameliorate neuronal death in several in vitro and in vivo models of neurodegenerative disease. However, delivery of polypeptide growth factors to compromised neurons in the CNS is problematic as the blood-brain barrier prevents systemic delivery, and chronic in-dwelling cannulae are required for intraparenchymal delivery. To circumvent these problems and specifically target neurotrophic factors to the environment surrounding degenerating neurons in the CNS, we have generated replication-defective adenovirus (Ad) vectors that contain a secretable form of ciliary neurotrophic factor (sCNTF) or neurotrophin-3 (NT-3). In this study, we demonstrate that sCNTF/Ad and NT-3/Ad can efficiently infect primary astrocytes, resulting in gene transcription and the production of functional protein. Using Northern blot analysis, dose-dependent expression of sCNTF or NT-3 mRNA was detected 7 days after infection. The levels of mRNA expressed in transgenic astrocytes was dependent on virus titer and increased with increasing virus concentration. sCNTF or NT-3 protein was also detected in astrocyte supernatants by immunoblot analysis and 2-site ELISA. ELISA indicated that astrocytes infected with sCNTF/Ad or NT-3/Ad secreted neurotrophic factors at a rate of approximately 120 pg/10(6) cells/h and 350 pg/10(6) cells/h, respectively. To test for secretion of bioactive sCNTF or NT-3 protein, E8 chick ciliary ganglion or nodose ganglion neurons were grown in medium conditioned by control astrocytes or astrocytes treated with sCNTF/Ad or NT-3/Ad, showing a robust and dose-dependent increase in neuronal survival when compared to control supernatant. In addition, motor neurons plated onto astrocyte monolayers pretreated with sCNTF/Ad showed a two- to fourfold increase in ChAT activity when compared to those grown on astrocytes pretreated with Lac-Z/Ad. This study demonstrates that, using replication-defective adenovirus, primary astrocytes can be efficiently engineered to secrete bioactive sCNTF or NT-3, resulting in enhanced survival of responsive peripheral and central neuronal populations.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Adenoviridae , Astrocitos/virología , Vectores Genéticos , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Northern Blotting , Supervivencia Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/metabolismo , Células Cultivadas/virología , Factor Neurotrófico Ciliar , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/genética , Genes myc/genética , Inmunohistoquímica , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Neuronas/virología , Neurotrofina 3 , Ratas , Proteínas Recombinantes/genética , Replicación ViralRESUMEN
Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that mediates survival and differentiation of neurons as well as many other cell types. In this study, CNTF and leukemia inhibitory factor (LIF) reduced the apparent number of primary serotonergic neurons in E14 raphe culture by 90% as determined by immunocytochemistry for serotonin (5HT). The reduction in 5HT cell number was not due to neuronal loss as removal of CNTF after 4 days in culture resulted in a partial restitution of the serotonergic phenotype. In the RN46A serotonergic cell line which is induced to become serotonergic by brain-derived neurotrophic factor (BDNF), the addition of CNTF suppressed tryptophan hydroxylase and 5HT synthesis and increased choline acetyl transferase (ChAT) expression by 6-fold and ChAT activity by 20- to 30-fold over 12 days. As with the primary neurons, removal and replacement of CNTF with BDNF after 4 days resulted in a partial restitution of 5HT expression. Moreover, other members of the CNTF-cytokine family that use gp130 and/or LIF receptor beta as their signal transducing receptors-LIF, oncostatin M, interleukin 6, and interleukin 11-had similar effects on increasing ChAT activity and reducing 5HT expression in RN46A cells. Analysis of 5HT levels showed no significant difference in the amount of serotonin between wild-type and CNTFR alpha knockout mice at birth, suggesting that the potential to switch phenotype mediated through CNTFR alpha is a latent property of neuroepithelial precursors in the raphe nucleus.
Asunto(s)
Fibras Colinérgicas/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Núcleos del Rafe/citología , Serotonina/fisiología , Acetilcolinesterasa/análisis , Acetilcolinesterasa/biosíntesis , Animales , Canales de Calcio/fisiología , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/química , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Cromatografía Líquida de Alta Presión , Factor Neurotrófico Ciliar , Inmunohistoquímica , Ratones , Ratones Noqueados , Neuronas/química , Neuronas/citología , Fenotipo , Ratas , Ratas Sprague-Dawley , Rombencéfalo/citología , Serotonina/biosíntesis , Serotonina/genética , Triptófano Hidroxilasa/análisis , Triptófano Hidroxilasa/genéticaRESUMEN
Neurotrophic factor expression in the adult mammalian CNS is largely neuronal. However, upon traumatic injury reactive astrocytes express a number of neurotrophic factors including ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), and NGF. In this study, we examined whether the upregulation of neurotrophic factors in reactive astrocytes and cultured astrocytes is a consequence of separation from their neuronal counterparts, and whether neurotrophic factor levels can be regulated by placing astrocytes into coculture with neurons. We show that reintroduction of rat hippocampal neurons to rat hippocampal astrocytes in vitro leads to a time dependent downregulation in astrocytes of the neurotrophic factors CNTF, NGF, and neurotrophin 3 (NT-3). In contrast, brain-derived neurotrophic factor (BDNF) mRNA, which is only expressed in neurons in these cultures is slightly increased. Once neurotrophic factor levels in cocultures had reached a steady state in the neuron/glia cocultures, we initiated a traumatic event with the excitotoxin kainic acid. BDNF protein was rapidly upregulated within 24 hr after lesion, whereas CNTF protein upregulation was delayed reaching maximal levels by 3 d. Despite the endogenous upregulation of both of these trophic factors, no activation of their respective receptors, as measured by tyrosine phosphorylation, was detectable following kainate administration. However, following addition of exogenous CNTF at any time point up to 24 hr after kainate administration, the beta components of the CNTF receptor (LIFR beta and gp130) could be phosphorylated. Furthermore, although activation of neuronal LIFR beta and gp130 by exogenous CNTF declined during the period of neuronal death, these receptors reappeared on astrocytes and could be activated by CNTF. In contrast, phosphorylation of TrkB by exogenous BDNF was undetectable by 24 hr and could not be reactivated after this point. These data suggest that the intimate association of astrocytes and neurons in the CNS serves to suppress astrocyte-derived neurotrophic factor expression and that neuronal loss leads to a derepression of neurotrophic factor synthesis in astrocytes. However, the upregulation of endogenous BDNF and CNTF observed after excitotoxic lesion in this culture model are insufficient to activate signal transduction and protect against neuronal loss.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Kaínico/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Astrocitos/ultraestructura , Muerte Celular , Técnicas de Cocultivo , Hipocampo/citología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Ratas , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factores de TiempoRESUMEN
Programmed death and the identification of growth factors delaying this process in the oligodendrocyte lineage suggest that other cell types provide oligodendrogliotrophins. To determine their source, primary cultures of oligodendroblasts immunopurified from postnatal rat cerebrum were used to screen other cultured neural and non-neural cell types for the release of survival factors into a defined insulin-containing medium. In non-conditioned medium, oligodendroblasts died 1-2 days after undergoing terminal differentiation into oligodendrocytes, as defined by the onset of expression of galactocerebroside. In medium conditioned by astrocytes, unlike the other tested cell types, differentiated oligodendrocytes survived for weeks in a mature myelinogenic state. Survival was partially reduced by immunoabsorption of the medium with antibodies to platelet-derived growth factor and abolished by immunoabsorption with antibodies to leukemia inhibitory factor. By the same criterion, survival activity was not attributed to other astrocytic products, ciliary neurotrophic factor and basic fibroblast growth factor. Membrane ultrafiltration analysis indicated the activity corresponded to heat-labile protein smaller (M(r) = 10(-30) x 10(3)) than native rat leukemia inhibitory factor (M(r) = 43 x 10(3)). The astrocytic stimulus was > 4-fold more efficacious than other known oligodendrogliotrophic cytokines, including ciliary neurotrophic factor, neurotrophin-3 and leukemia inhibitory factor itself, tested singly or in combination, and promoted survival additively with these agents. These findings suggest that astrocytes function as paracrine regulators of oligodendroblast and oligodendrocyte survival and that their effect is mediated initially by platelet-derived growth factor and thereafter by a powerful cytokine related to leukemia inhibitory factor.
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Astrocitos/fisiología , Inhibidores de Crecimiento/metabolismo , Interleucina-6 , Linfocinas/metabolismo , Oligodendroglía/citología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Astrocitos/citología , Comunicación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Medios de Cultivo Condicionados , Citocinas/metabolismo , Factor Inhibidor de Leucemia , Microscopía Fluorescente , RatasRESUMEN
Until recently, astrocytes were not considered as sites for neurotrophic factor action. We show here that, both in vivo and in vitro, astrocytes express receptors for two separate families of neurotrophic factors. In the intact adult rat CNS, astrocytes express the extracellular domain of the neurotrophin receptor TrkB and, in a more restricted population, the low-affinity nerve growth factor receptor p75LNGFR. In the lesioned CNS, expression of the alpha component of the receptor for ciliary neurotrophic factor (CNTFR alpha) switches from a purely neuronal localization to cells in the glial scar at the edge of the wound. Using cultured hippocampal astrocytes as a model to address the functional status of these receptors, we have found only the truncated forms of TrkB and TrkC, which are incapable of signal transduction as measured by protein tyrosine phosphorylation or immediate early gene induction. In contrast, a fully functional CNTF receptor complex capable of signal transduction is present on cultured astrocytes. Thus, the neurotrophin receptors may act primarily to sequester or present the neurotrophins, whereas in the case of CNTF a functional response can be initiated within the astrocyte.
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Astrocitos/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores de Factores de Crecimiento/fisiología , Animales , Células Cultivadas , Factor Neurotrófico Ciliar , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/efectos de los fármacos , Interleucina-6/farmacología , Factores Inhibidores de la Migración de Leucocitos/farmacología , Masculino , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor de Factor Neurotrófico Ciliar , Receptor trkC , Receptores de Factor de Crecimiento Nervioso/fisiología , Proteínas Recombinantes/biosíntesis , Transducción de Señal , Activación TranscripcionalRESUMEN
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine which is detectable only at very low levels in the intact adult rat CNS, but following an aspirative lesion to the dorsal hippocampus and overlying cortex, CNTF mRNA levels are dramatically up-regulated in reactive astrocytes. In cultured rat hippocampal astrocytes, CNTF mRNA levels are high, similar to the levels in reactive astrocytes in vivo, but are strongly suppressed after administration of isoproterenol and forskolin, which stimulate the production of intracellular cyclic AMP, induced marked morphological change in the astrocytes and up-regulate glial fibrillary acidic protein mRNA and nerve growth factor mRNA in these cells. Following a single administration of forskolin to cultured astrocytes, suppression of CNTF mRNA was sustained for up to 7 days. A similar down-regulation was observed with the endogenous adrenergic agonists noradrenaline and adrenaline as well as, to a lesser extent, dopamine and adenosine. Down-regulation of CNTF mRNA resulted in a gradual reduction in the level of CNTF protein within the astrocytes. A single addition of forskolin or isoproterenol resulted in a drop in CNTF protein levels to 29 and 52% of control levels respectively after 9 days in vitro, although the rate of turnover of CNTF remained the same. Down-regulation of CNTF mRNA in cultured hippocampal astrocytes by adenylyl cyclase activation was quite specific, as a wide range of growth factors, cytokines and neurotransmitters had little or no effect upon CNTF mRNA levels.
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Astrocitos/metabolismo , Hipocampo/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , ARN Mensajero/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Northern Blotting , Células Cultivadas , Factor Neurotrófico Ciliar , Colforsina/farmacología , AMP Cíclico/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/farmacología , Isoproterenol/farmacología , Cinética , Proteínas del Tejido Nervioso/análisis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Factores de TiempoRESUMEN
Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up-regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF-expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF-like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res., 553, 291-299, 1991), does not contribute appreciably to this trophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)